Polycyclic aromatic hydrocarbons (PAHs) require stereoselective activation by the cytochromes P450 (P450) and epoxide hydrolase (EH) to exert their mutagenicity and carcinogenicity. In this study, we utilize the cDNA expressed P450s and EH to determine their specificity in regio- and stereoselective metabolism of potent PAHs. In the metabolism of 7,12-dimethyl-benz[a]anthracene (DMBA), 15 products were identified. Of nine human P450s, 1A1 showed the highest activity (6- to 33-fold greater than any other P450s), and 1A2, 2B6 and 2C9 had moderate activities. The 30% and 15% contributions of 3A3/4/5 and 2E1 to total metabolism of DMBA in human liver were assessed by inhibitory monoclonal antibodies. The hydration kinetics (Km and Vmax) and enantiomeric preferences (Vmax/Km) indicate that EH favors RS-epoxides of chrysene (CR, 8.1-fold), dibenz[a,h]anthracene (DB[a,h]A, 7.8) and SR-epoxides of benzo[a]pyrene (B[a]P, 2.0), 3-methylcholanthrene (3MC, 13.8) and DMBA (5.7) over their respective enantiomers. EH preferentially opens the S C-O bond of R,S- epoxides of B[a]P (76%), 3MC (89%), CR (92%) and DB[a,h]A (96%) and the S C-O bond of S,R-epoxides of B[a]P (95%) and DMBA (85%). Regression analyses by a 10-ring PAH template suggest that interaction with four rings are important in determining the stereoselectivity of the EH. These results provide information on the binding interaction and kinetics of EH and help us understand the role of EH in overall stereoselective metabolism of PAHs. Since seven human, five rodent and two bacterial P450s all convert B[a]P to the potent stereoisomer, the crystal structures of bacterial P450s were used to hypothesize the stereochemical outcome of B[a]P metabolism. Strikingly similar results were observed when B[a]P was docked in the active site of enzymes. Molecular mechanics is used to determine the specific amino acids responsible for the steric effect on substrate. Molecular dynamics indicates that a single helical region, one that is likely to be conserved in all P450s, plays a primary role in determining the stereoselectivity of the reaction.